TYROSINE MODIFICATION BY REACTIVE NITROGEN SPECIES - A CLOSER LOOK

Peroxynitrite (ONOO-) is a powerful oxidant and cytotoxic species formed by the rapid reaction between nitrogen monoxide (nitric oxide, (NO)-N-.) and superoxide (O-2(.-)). At neutral pH ONOO- is partly protonated and this protonated form, peroxynitrous acid (ONOOH), decomposes rapidly to nitrate, forming (an) intermediate(s) with reactivity similar to (OH)-O-. and (NO2)-N-.. Peroxynitrite can hydroxylate and nitrate aromatic rings, and aromatic nitration of phenols such as tyrosine by ONOOH is proposed to proceed via a radical mechanism, with intermediate formation of (NO2)-N-.. Modification of tyrosine by (NO2)-N-. also involves nitration via a radical mechanism. Aromatic nitration of phenols by ONOO- has been shown to be enhanced by superoxide dismutase or Fe3+-EDTA, which were proposed to catalyze heterolytic cleavage of ONOOH to form a nitrating species similar to the nitronium ion (NO2+). We investigated possible mechanisms of tyrosine modification by various reactive nitrogen species, including ONOO-, 3-morpholinosydnonimine (SIN-1), and (NO2)-N-.. Reaction of tyrosine with ONOO- leads to formation of 3-nitrotyrosine and dityrosine, indicating intermediate formation of tyrosyl radicals. The pH dependence of formation of both 3-nitrotyrosine and dityrosine by ONOO- suggests that intermediate formation of ONOOH is required. Qualitatively similar results were obtained when ONOOH was generated continuously by H2O2 and NaNO2 at mildy acidic pH or with SIN-1, a compound which at neutral pH releases both (NO)-N-. and O-2(.-), presumably producing ONOO-. However, relatively low yields of nitrotyrosine were obtained with SIN-1, possibly because of competing reactions of tyrosyl radicals with (NO)-N-. or O-2(.-). Possible involvement of (NO2)-N-. in tyrosine modification by ONOO- was studied using hydroxyl radical scavengers, which can increase the radical yield during decomposition of ONOOH and thereby enhance generation of (NO2)-N-.. Hydroxyl radical scavengers did not affect tyrosine modification by (NO2)-N-. directly and slightly inhibited tyrosine modification by authentic ONOO-. However, when ONOO- was produced at a slower rate, either by SIN-I or by H2O2/NaNO2 at acidic pH, hydroxyl radical scavengers were found to significantly enhance tyrosine nitration. Our results suggest that ONOO- or ONOO--generating systems induce nitration of tyrosine (or tyrosine residues in proteins) via intermediate formation of tyrosyl radicals and (NO2)-N-.. (C) 1995 Academic Press, Inc.